Fish & Shellfish Immunology (2008) 25, 128e136 available at www.sciencedirect.com journal homepage: www.elsevier.com/locate/fsi Studies on Bacillus subtilis and Lactobacillus acidophilus, as potential probiotics, on the immune response and resistance of Tilapia nilotica (Oreochromis niloticus) to challenge infections Salah Mesalhy Aly a,*, Yousef Abdel-Galil Ahmed b, Ahlam Abdel-Aziz Ghareeb b, Moahmed Fathi Mohamed a a b Department of Fish Health, WorldFish Center, Regional Research Center for Africa and West Asia, Abbassa, Sharkia, Egypt Department of Bacteriology, Mycology and Immunology, Faculty of Veterinary Medicine, Zagazig University, Egypt Received 14 February 2008; revised 17 March 2008; accepted 18 March 2008 Available online 28 March 2008 KEYWORDS Probiotics; Bacillus subtilis; Lactobacillus acidophilus; Immune parameters; Aeromonas hydrophila; Pseudomonas fluorescens; Streptococcus iniae; Oreochromis niloticus Abstract The probiotic activity of two bacteria (Bacillus subtilis and Lactobacillus acidophilus) was evaluated by its effect on the immune response of Nile tilapia (Oreochromis niloticus), beside its protective effect against challenge infections. Furthermore, their in-vitro inhibitory activity was evaluated. The in-vitro antimicrobial assay showed that Bacillus subtilis and Lactobacillus acidophilus inhibited the growth of A. hydrophila. The B. subtilis inhibited the development of P. fluorescens while L. acidophilus inhibited the growth of Strept. iniae. The B. subtilis and L. acidophilus proved harmless when injected in the O. niloticus. The feed, containing a mixture of B. subtilis and L. acidophilus or B. subtilis alone, showed significantly greater numbers of viable cells than feed containing L. acidophilus only after 1, 2, 3 and 4 weeks of storage at 4  C and 25  C. The survival rate and the body-weight gain were significantly increased in the fish given B. subtilis and L. acidophilus for one and two months after application. The hematocrit values showed a significant increase in the group that received the mixture of B. subtilis and L. acidophilus compared with the control group. The nitroblue tetrazolium (NBT) assay, neutrophil adherence and lysozyme activity, showed a significant increase in all the probiotic-treated groups after 1 and 2 months of feeding, when compared with the untreated control group. The serum bactericidal activity was high in the group that was given a mixture of the two bacteria. * Corresponding author. Department of Bacteriology and Immunology, WorldFish Center, Regional Research Center, Abbassa, Sharkia, Egypt. Tel.: þ20 55 340 4228; fax: þ20 55 340 5578; Mobile: þ20 12 105 7688. E-mail address: s.mesalhy@cgiar.org (S.M. Aly). 1050-4648/$ - see front matter ª 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2008.03.013 Studies on Bacillus subtilis and Lactobacillus acidophilus, as potential probiotics 129 The relative level of protection (RLP) was significantly higher against A. hydrophila, in the bacterial mixture treated group and against P. fluorescens in the L. acidophilus treated group, after one month of the feeding trial. A significantly higher RLP, against A. hydrophila or P. fluorescens, was noticed after 2 months of the feeding trial in the group given a mixture of the two bacteria, and against Strept. iniae in the group fed a diet containing L. acidophilus. ª 2008 Elsevier Ltd. All rights reserved. Introduction Aquatic animals in large-scale production facilities are exposed to stress conditions, diseases and deterioration of the environmental conditions, leading to serious economic losses [1,2]. The prevention and treatment of the infectious aquatic animal-diseases, in Egypt, include a limited number of Government-approved antibiotics and chemotherapeutics, beside limited vaccines that can be used to assist the environmental management. However, the use of antibiotics can lead to the development of antibiotic-resistant bacterial strains [3] and may modulate the immune response [4]. A promising alternative approach for controlling fish diseases is the use of probiotics or beneficial bacteria, which control pathogens through a variety of mechanisms. The use of probiotics, in human and animal nutrition, is well documented [5] and recently, have been applied to aquaculture [6,7]. Bacillus subtilis (B. subtilis) has been shown to possess antitumor and immunomodulatory activities [8]. Some studies have demonstrated that B. subtilis and spores of B. subtilis act as probiotics by promoting the growth and viability of the beneficial lactic acid bacteria in the intestinal tracts of humans and some animals [9]. The Lactobacillus acidophilus (L. acidophilus) has been considered to be the predominant lactobacillus in the intestinal tract of healthy humans [10]. L. acidophilus strains have been widely utilized as a dairy starter culture for their therapeutic activities associated with an intestinal microbial balance. Probiotics are defined as cultures of live microorganisms that benefit the host (humans and animals) by improving the properties of the indigenous microflora [11]. Such effects have been attributed to biochemical, physiological, and antimicrobial effects, as well as competitive exclusion in the intestinal tract [12]. The present study aimed to evaluate the efficiency of using two bacteria (Bacillus subtilis and Lactobacillus acidophilus) as a potential probiotic in the farming of Nile tilapia (Oreochromis niloticus). The evaluation was based upon their safety, in-vitro inhibitory activity, and effects on the immune response. Moreover, the survival rate and growth performance were considered, besides the possible protective effects against a challenge infection. Material and methods Fish Two-thousand and one hundred apparently healthy, Nile tilapia (O. niloticus) of both sexes were collected from the WorldFish Center, Abbassa, Egypt. One hundred and eighty O. niloticus (65  5 g) were used to test the safety of the used probiotic strains. The remaining 1920 O. niloticus (5  1.3 g) were used for the feeding experiment. They were kept for 2 weeks under observation for acclimatization in glass aquaria (60  50  70 cm). The water of the aquaria was renewed daily, and its temperature was maintained at 26  1  C. Bacterial strains The probiotic, Bacillus subtilis (B. subtilis) (ATCC 6633) was obtained as lyophilized cells from Sigma. Lactobacillus acidophilus (L. acidophilus) was kindly supplied as a reference strain from the Animal Health Research Institute, Dokki, Egypt. The pathogenic strains, Aeromonas hydrophila, Pseudomonas fluorescens and Streptococcus iniae were obtained, as reference strains, from the Fish Health Laboratory at The WorldFish Center, Abbassa, Egypt. In-vitro antimicrobial activity assay (Agar spot assay) The probiotic strains (B. sublitis and L. acidophilus) were cultured in Trypticase soya broth for 24 h at 30  C. Spots were then made by pouring 10 ml of overnight cultures of B. sublitis and L. acidophilus, each on one side of the trypticase soya agar plates. The plates were incubated overnight at 30  C and the growth of the strains was checked the following day. After the spot development, a soft agar (composed of Tryptone Soya Broth þ0.7% bacteriological agar, containing 5% of overnight cultures of the pathogenic strain from each of A. hydrophila, P. fluorescens and Strept. iniae in tryptone soya broth) was poured on the plates. The inhibition was recorded by measuring the absence of pathogen growth around the spots. All tests were performed in duplicate [13]. Safety of probiotic strains One hundred and eighty tilapia (65  5 g) were divided into 3 equal groups (60 fish) in three replicates (each of 20 fish) and distributed randomly among 9 aquaria. The first group was intraperitoneally (I/P) injected with 0.5 ml L. acidophilus fresh culture suspension containing 107 bacteria ml1 while the second group was I/P injected with 0.5 ml B. subtilis fresh culture suspension containing 107 bacteria ml1. The third group served as a control and I/P injected with 0.5 ml sterile saline (0.85% NaCl). Both the test and control groups of fish were observed and fed on a basal diet containing 30% protein and water temperature was 26  1  C throughout the experiment. The mortality rate was recorded daily for 15 days. 130 Determination of the survival of the probiotics bacteria in feed The dietary ingredients were obtained from specialized factories and prepared locally in pelleted form. The basal diets were prepared by grinding and sieving the corn to granules of 0.5 mm (Thomes-Willey Laboratory Mill Model 4). The ingredients were mixed mechanically in a horizontal mixer (Hobarts model D300T, Troy, OH, USA) at a low speed for 30 min. The oil (corn & cod liver) was added gradually to assure the homogeneity of the ingredients. The mixing speed was increased for 5 min during the addition of water (12% moisture) until clumps began to form. The mixture was sterilized and the pellets (0.5 cm diam) were prepared using a pellet-machine (CPM California Pellet Mill Co., San Fransisco, CA, USA). The pellets were left for 24 h to dry under aseptic conditions. The probiotic bacteria (B. subtilis and/or L. acidophilus) were prepared by the inoculation of the bacterial isolates in TSB and incubated at 30  C for 48 h. The cultures were centrifuged (Beckman, Alaska, HI, USA) at 3000 rpm for 30 min. The pellets were washed twice with saline. The bacteria were counted. Three probiotic-supplemented diets were prepared. Diet (1) was mixed with 0.5  107 L. acidophilus and 0.5  107 B. subtilis/g diet. Diet (2) was mixed with 1  107 L. acidophilus/g diet. Diet (3) was supplemented with 1  107 B. subtilis/g diet. The survival of the supplemented bacteria in the diet was assessed following storage at 4  C and at room temperature (25  C) for four weeks. One gram of the diet was weekly homogenized in 9.0 ml saline, and serial dilutions down to 104 were prepared and 0.1 ml was spread onto triplicate plates of tryptic soya agar. The colonies were counted after incubation for 24 h at 30  1  C as described by Irianto and Austin [6]. S.M. Aly et al. were determined at the end of the 4th and 8th weeks of the experiment. A number of hematological and immunological tests were made as well as challenge tests. Survival rate The fish were counted after 4 and 8 weeks from the start of the experiment to determine the survival percentage: Survival % Z (No. of fish counted/No. of stocked fish)  100. Body weight gain and feed conversion rate The body weight gain of fish was determined as the difference between the initial and final weights at the ends of one and two months from the start of the experiment. Feed conversion rate was calculated according to the following formula: FCRZðwf  wi=F Þ  100 Where: wf Z final weight of fish (g), wi Z initial weight of fish (g) & F Z amount of feed (g). Blood sampling Twenty fish were randomly collected from each treatment and the control. The fish were anesthetized by immersion in water containing 0.1 ppm tricaine methane sulfonate (MS222). Whole blood (0.5 ml) was collected from the caudal vein of each fish using syringes (1-ml) and 27-gauge needles that were rinsed in heparin (15 unit ml1), to determine the hematocrit values, NBT, and neutrophil adherence tests. A further 0.5 ml blood-sample was centrifuged at 1000  g for 5 min in order to separate the plasma. The latter was stored at 20  C to be used for lysozyme activity test. For separation of serum, blood samples (0.5 ml) were withdrawn from the fish caudal vein, as before, and transferred to Eppendorf tubes without anticoagulant. The blood samples were centrifuged at 3000  g for 15 min and the supernatant serum was collected and stored at 20  C until used for the serum bactericidal test. Feeding and challenge experiment One thousand nine hundred and twenty fingerlings were divided into four equal groups, each of 480 fish. The groups were subdivided into 16 equal subgroups of 30 fish (mean body weight 5.2  0.9 g) to determine the probiotic-protective effect against challenge. The fingerlings were allocated in 64 aquaria (60  70  50 cm) containing 150 L of water. The basal diet was fed to all fish during the week of acclimatization. The water was renewed daily. Low-pressure electric air pumps provided aeration via air stones and dissolved oxygen (DO) levels was maintained at or near the saturation levels. Water temperature was 26  1  C throughout the trial. The 1st group was fed on diet supplemented with L. acidophilus (0.5  107 bacteria g1 diet) and B. subtilis (0.5  107 bacteria g1). The 2nd group was fed on diet supplemented with L. acidophilus (1  107 bacteria g1). The 3rd group was fed on diet incorporated with B. subtilis (1  107 bacteria g1). The 4th group was given basal diet without probiotics (control). The fish were daily fed at a rate of 5% of the body weight for 8 weeks. All the diets were prepared twice a week and stored at 4  C. The weight of all fish in each aquarium was obtained weekly and the feed ratios were adjusted accordingly. The survival rate and the gain in the body weight Hematocrit level Hematocrit capillary tubes were two-third filled with the whole blood and centrifuged in a hematocrit centrifuge for 5 min and the percentage of the packed cell-volume was determined by the hematocrit tube reader [14]. Nitroblue tetrazolium activity (NBT) Blood (0.1 ml) was placed in microtiter plate wells, to which an equal amount of 0.2% NBT solution was added and incubated for 30 min at room temperature. A sample of NBT blood cell suspension (0.05 ml) was added to a glass tube containing 1 ml N,N-dimethyl formamide and centrifuged for 5 min at 3000 rpm. The supernatant fluid was measured in a spectrophotometer at 620 nm in 1 ml cuvettes [15]. Adherence/NBT assays NBT-glass adherent assays were performed by placing single drops of blood (0.1 ml) on 2 glass coverslips and incubating them for 30 min at room temperature. The coverslips were then gently washed with phosphate buffered saline (PBS). Drops (0.1 ml) of 0.2% NBT were placed on microscope slides and covered by a coverslip, then incubated at room temperature for 30 min with the NBT solution. The Studies on Bacillus subtilis and Lactobacillus acidophilus, as potential probiotics activated neutrophils were microscope (400) [16]. then counted under a Lysozyme activity The lysozyme activity was measured using the turbidity assay. Chicken egg lysozyme (Sigma) was used as a standard and 0.2 mg ml1 lyophilised Micrococcus lysodeikticus in 0.04 M sodium phosphate buffer (pH 5.75) was used as substrate. Fifty ml of serum was added to 2 ml of the bacterial suspension and the reduction in the absorbance at 540 nm was determined after 0.5 and 4.5 min incubation at 22  C. One unit of lysozyme activity was defined as a reduction in absorbance of 0.001 min1 [17]. Serum bactericidal activity (SBT) Bacterial cultures of A. hydrophila, P. fluorescens and Strept. iniae were centrifuged, and the pellet was washed and suspended in phosphate buffered saline (PBS). The optical density of the suspension was adjusted to 0.5 at 546 nm. This bacterial suspension was serially diluted (1:10) with PBS five times. The serum bactericidal activity was determined by incubating 2 ml of the diluted bacterial suspension with 20 ml of the serum in a micro-vial for 1 h at 37  C. PBS replaced the serum in the bacterial control group. The number of viable bacteria was determined by counting the colonies after culturing on trypticase soya agar plates for 24 h at 37  C [18]. Challenge test One month after the start of the feeding experiments, 180 fish were collected from each of the 3 treated and control groups and divided into three sub-groups, each of 60 fish that was then re-distributed equally among 3 aquaria. Fish from the 1st, 2nd and 3rd subgroup were challenged I/P with 0.5 ml of fresh culture suspension containing 108 bacteria ml1 A. hydrophila, P. fluorescens and Strept. iniae, respectively. The same challenge test was repeated 2 month later on the other 180 fish from each of the 4 groups. The challenged fish were kept under observation for 15 days and the dead fish were used for bacterial re-isolation. The mortalities were recorded and the relative level of protection (RLP) among the challenged fish was determined [19] using the following equation: 131 P. fluorescens of 10 and 7 mm, respectively, but no such changes were detected against Strept. iniae. The L. acidophilus caused an inhibition zone against A. hydrophila and Strept. iniae of 8 and 7 mm, respectively, without any visible inhibition zone against the P. fluorescens. The I/P injection of B. subtilis or L. acidophilus was harmless to the O. niloticus as neither mortalities nor morbidities were observed during the 15 days of observation. The storage of feed supplemented by either a mixture of B. subtilis and L. acidophilus or B. subtilis alone showed a statistically significant higher number of viable cells when compared with feed that was supplemented with L. acidophilus after 1, 2, 3 and 4 weeks of storage at both 4  C and 25  C. There was a significantly greater number of viable cells in the probiotic feed stored in the refrigerator at 4  C than that stored at room temperature (25  C) in all treatments (Table 1). The body weight gain was 22.92, 26.16, 25.46 & 14.20 after 1 month and 32.36, 35.69, 34.65 & 24.85 after 2 months for the group fed on B. subtilis & L. acidophilus, L. acidophilus, B. subtilis and only basal diet (control group); respectively. The feed conversion rate was 1.56, 1.58, 1.59 & 1.70 after 1 month and 1.69, 1.71, 1.71 & 1.82 after 2 months for the same groups respectively. The survival rate among the experimented fish, was 96.0, 90.0, 88.0 & 86.0 at the end of the first month and 93.0, 83.3, 78.0 & 78.2 at the end of second month for the group fed on B. subtilis & L. acidophilus, L. acidophilus, B. subtilis and only basal diet (control group); respectively (Graphs 1 and 2). The hematocrit values were significantly higher in the group that received the mixture of B. subtilis and L. acidophilus compared with the control group but no significant difference was observed among the probiotic treated groups or the fish at the end of the 1st and 2nd months after the inception of the feeding experiment. The NBT assay, neutrophil adherence and lysozyme activity, at 1 and 2 months of feeding were significantly higher in all groups given the probiotic-supplemented diet when compared with the untreated control group. There were no statistically significant differences between the results after 1 and 2 months or among the probiotic groups within the same period, with the exception of the NBT in the group of mixed bacteria (Table 2). RLPZ1  ðpercent of mortality in treated groupOpercent of mortality in control groupÞ  100: Statistical analysis Analysis of Variance (ANOVA) and Duncan’s multiple Range Test [20] was used to determine the differences between treatments. The mean values were significant at the level of (P < 0.05). Standard errors, of treatment-means, were estimated. All the statistics were carried out using Statistical Analysis Systems (SAS) program [21]. Results The in-vitro antimicrobial assay showed that the B. subtilis induced an inhibition zone against A. hydrophila and The serum bactericidal activity, in all the probiotic treated groups against A. hydrophila, P. fluorescens and Strept. iniae, was significantly higher than in the untreated control group. However, the group which was given a mixture of B. subtilis and L. acidophilus showed higher bactericidal activity than the other treated groups. The number of the bacterial colonies, in the group which was given the mixture of B. subtilis and L. acidophilus, was significantly lower at 2 months than that at 1 month after the commencement of the feeding trial, but the other groups revealed inconsistent responses (Table 3). The RLP against A. hydrophila, P. fluorescens and Strept. iniae, in the group fed on diet supplemented with 132 S.M. Aly et al. Table 1 Viability of the probiotics, in the supplemented diets, after storage at 4  C and 25  C (mean  standard error) Period B. subtilis & L. acidophilus in 4 C 25  C day 0 7th 14th 21st 28th L. acidophilus  B. subtilis  4 C 4 C 25 C 7.582Aa  0.374 7.582Aa  0.374 7.528Aa  0.619 7.528Aa  0.619 Aa Ab Ba 1.782  0.417 2.224  0.557 0.672Bb  0.160 4.372  0.391 Aa Ab Ba 1.111  0.122 0.0792  0.018 0.065  0.022 0.0116Bb  0.004 0.025Aa  0.007 0.0013Ab  0.0004 0.0002Ba  0.00004 0.0009Ba  0.0005 0.0016Aa  0.0004 0.0003Ab  0.00016 0.00008Ba  0.00006 0.00004Bb  0.00008 25  C 7.844Aa  0.559 7.84Aa  0.59 Aa 4.624  0.460 1.88Ab  0.161 Aa 0.98  0.099 0.071Ab  0.0123 0.028Aa  0.003 0.0015Ab  0.0013 0.001Aa  0.0008 0.00036Ab  0.0001 Upper case letter-superscripts denote significant differences among the treatments, within the same period. Lower case letterssuperscripts denote significant differences among the different periods within the same treatment. B. subtilis and L. acidophilus mixture, was statistically significantly higher than in the groups that were given either B. subtilis or L. acidophilus alone at the end of the 1st and 2nd months after the start of the trial. The RLP, for each probiotic group, was high at the end of the 2nd month than the 1st month. The significance of the increase varied with the type of the probiotic used and the period of feeding (Table 4). Discussion Antimicrobial activities were induced by B. subtilis against A. hydrophila and P. fluorescens, but not against Strept. iniae in this study. A similar inhibitory activity for Bacillus subtilis and Bacillus S11 strain were demonstrated previously [22,23]. Such antimicrobial activity could be attributed to the fact that the bacillus bacteria can be stimulated to compete with other fast growing bacteria for nutrients [24]. This phenomenon has been exploited for the production of polymyxin, bacitracin and gramicidin antibiotics from bacilli [25,26]. It is evident that the 40 a inhibitory mechanisms of probiotics differ as the L. acidophilus induced inhibition zones against A. hydrophila and Strept. iniae, but not against P. fluorescens. Groups of lactic acid bacteria (LAB) have been reported to exhibit, in-vitro, inhibitory activities against Gram-positive and Gram-negative fish pathogens [27e30]. Moreover, the inhibitory mechanism, induced by the Lactobacillus isolates was acid production [13]. Both B. subtilis and L. acidophilus, were judged to be safe and harmless, in the current work. The present finding agreed with Austin et al. [31] who proved the safety of a number of probiotics via I/M and I/P injection of Atlantic salmon. The storage of the probiotic-supplemented diet, under cold temperature demonstrated the durability of B. subtilis and L. acidophilus together or B. subtilis alone in the feed. On the other hand, Irianto and Austin [6] found that the probiotic-activity declined over an eight week period, when incorporated in the diets. The fish that received a mixture of the B. subtilis and L. acidophilus showed significantly higher survival rate than in the untreated control group. Similar findings were reported by others [32,33] where feed containing Bacillus spp. and Bacillus S11 increased survival rate of channel a ab 35 a 100 30 a 90 a b a b b 25 b b b b 80 ab 70 20 60 b 50 15 40 10 30 5 20 10 0 one month Two months 0 one month B. subtilus & L. acidophilus L. acidophilus B. subtilus Control Graph 1 Body weight gain of O. niloticus after feeding probiotic-supplemented diets for 1 and 2 months. Columns with the same letter are not significantly different. B. subtilus & L. acidophilus L. acidophilus Two months B. subtilus Control Graph 2 Survival percentage of O. niloticus after feeding probiotic-supplemented diets for 1 and 2 months. Columns with the same letter are not significantly different. Hematocrit value and some immunological tests of O. niloticus given probiotic-supplemented diet for 1 & 2 months (mean  standard error) Group/treatment One month Hematocrit (%) 1. B. subtilis & L. acidophilus 2. L. acidophilus 3. B. subtilis 4. Control Two months NBT mg/ml Lysozyme activity unit/ml Neutrophil adherence cell/field Hematocrit (%) NBT mg/ml Lysozyme activity unit/ml Neutrophil adherence cell/field 35.4Aa  1.12 2.13Aa  0.02 13.05Aa  0.53 13.13Aa  0.83 36.33Aa  0.99 2.22Aa  0.06 13.46Aa  0.51 13.62Aa  0.66 33.8ABa  1.48 33.29ABa  0.98 31.29Ba  0.85 1.95Ba  0.04 2.08ABa  0.06 1.77Ca  0.07 12.52Aa  0.59 12.65Aa  0.6 9.06Ba  0.3 12.67Aa  0.79 12.9Aa  0.7 7.8Ba  0.083 34.67ABa  1.31 35.8Aa  1.03 31.8Aa  1.06 2.08Ba  0.05 2.13ABa  0.04 1.85Ca  0.03 12.88Aa  0.52 12.99Aa  0.69 9.53Ba  0.59 13.07Aa  0.7 13.27Aa  0.09 8.79Ba  0.54 Twenty blood samples were randomly collected from each group. Upper case letter-superscripts denote significant differences among treatments during the same period. Lower case letters-superscripts denote significant differences among different periods within the same treatment. Table 3 Serum bactericidal activity of O. niloticus against pathogenic bacterial isolates after feeding probiotics for 1 & 2 months (bacterial count mean  standard error) Group/treatment One month A. hydrophila 1. 2. 3. 4. B. subtilis & L. acidophilus L. acidophilus B. subtilis Control Ba 366  16.31 413Ba  35.13 369Ba  16.62 564Aa  50.16 Studies on Bacillus subtilis and Lactobacillus acidophilus, as potential probiotics Table 2 Two months P. fluorescens Ca 370  22.8 470Ba  24.9 402BCa  43.75 584Aa  14 Strept. iniae Ba 401  15.84 404.8Ba  42.72 446.2Ba  18.11 597Aa  13.3 A. hydrophila Bb 243  16.93 287Ba  44 267Ba  27.8 518Aa  64.19 P. fluorescens Cb 264  31.63 370Bb  27.77 313BCb  45.34 557Aa  24.95 Strept. iniae 314Ba  15.33 291Ba  43.4 361Ba  32.97 552Aa  33.77 Twenty samples were randomly collected from each group. Upper case letter-superscripts denote significant differences among treatments within the same pathogen/period. Lower case letters-superscripts denote significant differences between the two periods within the same treatment/pathogen. 133 134 S.M. Aly et al. Table 4 Relative level of protection among O. niloticus after challenge infections at the end of 1st and 2nd months of feeding probiotic-supplemented diet Group/treatments One month (%) A. hydrophila 1. B. subtilis & L. acidophilus 2. L. acidophilus 3. B. subtilis 4. Control 36.84 Aa  3.83 33.3ABa  0.99 27.53Ba  3.78 0C Two months (%) P. fluorescens Strept. iniae A. hydrophila P. fluorescens Aa Aa Aa Ab 36.96  1.91 19.94Ba  4.76 31.06ABa  6.5 0C 32.88  5.24 29.98Aa  6.33 20.52Aa  5.89 0B 52.01  4.79 43.51Aa  7.12 48.32Ab  4.66 0B 51.18  5.85 33.27Bb  3.21 43.16ABa  5.55 0C Strept. iniae 40.56Ab  8.77 46.73Ab  5.09 26.86Aa  12.89 0B At 1 month experiment, 720 fish were used in 4 equal groups (180 each), each of three equal sub-groups (60, each), same numbers used at two months (total fish used 1440 fish). Upper case letter-superscripts denote significant differences between treatments within the same pathogen/period. Lower case letters-superscripts denote significant differences between the two periods with the same treatment pathogen. catfish (Ictalurus punctatus) and the shrimp Penaeus monodon. Moreover, the feeding of probiotic-supplemented diet (Lactobacillus fructivorans and Lactobacillus plantarum) increased the level of Ig and acidophilic granulocytes in the sea bream gut, stimulating the gut immune system, which correlated with improvements in the fry survival [34]. The currently used probiotic-supplemented diets increased the body weight gain, as a significant increase was encountered in the groups which were given B. subtilis or L. acidophilus than the untreated control. Such increase in the body-weight gain, in fish fed on probiotic-supplemented diets, could be attributed to the improved digestive activity by enhancing the synthesis of vitamins, cofactors and enzymatic activity [35e37], with a consequent improvement of the digestion, nutrient absorption and weight gain. Planas et al. [38] found that the addition of LAB increased the specific maximum growth rate of rotifer (Brachionus plicatilis). Carnevali et al. [39] noticed that, when the European sea bass was fed on Lactobacillus delbrueckii for 59 days, it showed 81% greater body weight and 28% greater weight gain after 25 days when compared with the control. The hematocrit values were increased with no statistically significant difference, among the treated groups. The increased value of the hematocrit, after 1 and 2 months of feeding, indicated the safety of the probiotics used and their efficacy in improving the health status as a reduced hematocrit can indicate that fish are not eating or are suffering infections [40]. The NBT test is used to determine the respiratory burst activity, especially of neutrophils and monocytes. The NBT test showed significantly increased values in all the current tested groups which were given probiotics when compared with the control group. The group that was given a mixture of the two probiotic-bacteria, showed higher values than the other groups. This suggests that the probiotics may enhance non-specific immune responses. The probiont including LAB increases the activities of phagocytes, lysozyme and complement [41,42]. The administration of Lactobacillus delbrüeckii sp. lactis and Bacillus subtilis, singly or in combination, increased phagocytic activity [43]. Dı́az-Rosales et al. [44] observed a higher phagocytic ability in fish given a mixture of two inactivated bacteria. However, no significant difference was noticed between the results of NBT after 1 and 2 months of feeding. The adherence test is an important early indicator of activated neutrophils and monocytes. Lysozyme has bactericidal activity and can be an opsonin that activates the complement system and phagocytes [45]. The lysozyme activity and neutrophil adherence test, in this study, showed a significant increase in all groups given probiotic-supplemented diets when compared with the untreated control group. There was insignificance difference between the probiotics-supplemented groups after 1 and 2 month of feeding. It has been shown that the injection of b-glucan induced a significantly elevated lysozyme activity [46]. The serum bactericidal activity (SBT), against the tested pathogens, was the highest in the group that was given the mixture of the two bacteria, especially after 2 month of application. The viable bacterial counts were lower in serum from all probiotic-treated groups, when compared with the control group. The decrease was significant in the case of A. hydrophila and Strept. iniae, and insignificant with P. fluorescens. Misra et al. [46] mentioned that the serum bactericidal activity, in fish injected with different dosages of b-glucan, was always significantly (P < 0.05) higher than in the control. The increased serum bactericidal activity in Achyranthes treated groups indicates that various humoral factors are involved in the innate and/or acquired immunities [18]. Similarly, Quil-A, a fraction from Quillaja saponaria Molina, enhanced the serum bactericidal activity in Oncorhynchus gairdneri Richardson [47]. The group that was fed the mixture of B. subtilis and L. acidophilus showed higher levels of protection against the test pathogens than the other groups and that fed for 2 months gave higher levels of protection than for 1 month. It was shown that the administration of Bacillus S11 or b-1,3-glucan or Lactobacillus significantly enhanced the survival of P. monodon yellowtail and turbot larvae, after challenge infections [33,48,49]. It could be concluded that potential probiotics can be used to enhance the immune and health status, thereby improving the disease resistance in O. niloticus and enhanced the growth performance. Application for one month was sufficient to improve the immune status, and a mixture of the two bacteria was superior. However, further extensive testing, including field and full commercial cost benefit analysis, is necessary before recommending its widespread application in aquaculture. Studies on Bacillus subtilis and Lactobacillus acidophilus, as potential probiotics Acknowledgements The authors thank Dr. Patrick Dugan, Dr. Malcolm Beveridge and all other colleagues at The WorldFish Center for their generous support without which this work would not have been possible. [19] [20] [21] References [1] FAO. The state of world fisheries and aquaculture. Rome, Italy; 2004. p. 14e17. [2] Subasinghe RP. 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